In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization

Bradley Hall1, Seyed Arshad2, Kyunghyun Seo2, Catherine Bowman2, Meredith Corley2, Sulay D. Jhaveri3, Andrew D. Ellington2

1 Department of Chemistry and Biochemistry, University of Texas, Austin, Texas, 2 Freshman Research Initiative, University of Texas, Austin, Texas, 3 Nova Research, Inc., Alexandria, Virginia
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 24.3
DOI:  10.1002/0471142727.mb2403s88
Online Posting Date:  October, 2009
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Abstract

This unit describes the selection of aptamers from a pool of single‐stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription. Curr. Protoc. Mol. Biol. 88:24.3.1‐24.3.27. © 2009 by John Wiley & Sons, Inc.

Keywords: aptamer; in vitro selection; affinity reagent; filter binding assay; SELEX

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Transcription and Isolation of RNA Pools
  • Support Protocol 1: Radiolabeling RNA for Use in an Initial Affinity Assay
  • Support Protocol 2: Binding Assay with the End‐Labeled RNA Pool to Determine the Optimal Protein Concentration for Selection
  • Basic Protocol 2: Isolating a Functionally Enriched Pool of RNA
  • Basic Protocol 3: Amplifying Selected Protein‐Binding RNA Species
  • Support Protocol 3: Assaying the Accumulation of Protein‐Binding RNA Species
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transcription and Isolation of RNA Pools

  Materials
  • Double‐stranded DNA pool (unit 24.2)
  • High Yield AmpliScribe T7 In Vitro Transcription Kit (Epicentre)
  • 8% polyacrylamide denaturing gel (see recipe and unit 2.12)
  • 2× denaturing dye (see recipe)
  • TBE buffer ( appendix 22)
  • TE buffer, pH 8.0 (see recipe)
  • 3 M sodium acetate ( appendix 22)
  • 70% and 95% ethanol
  • Thermal cycler, incubators, or heat blocks set at 37° or 42°C (for transcription) and 65° to 75°C (for denaturation)
  • UV light source
  • Fluorescent TLC plate (VWR) wrapped in plastic wrap
  • Sharp razor blade, fresh or thoroughly cleaned
  • Spectrophotometer, such as a NanoDrop (Thermo Scientific)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (unit 2.12)
NOTE: All solutions and buffers should be made with deionized water (18.2 MΩ resistivity), filtered through a 0.2‐µm polyethersulfone (PES) membrane, and sterilized by autoclaving. Use sterile, disposable plasticware and micropipet tips with filter barriers where possible. See Chapter 4 introduction and unit 4.1 for guidelines on standard methods to protect against contaminating RNases. If ribonuclease contamination is found to occur, it may be eliminated by treating water with diethylpyrocarbonate (DEPC; see unit 4.1).

Support Protocol 1: Radiolabeling RNA for Use in an Initial Affinity Assay

  Materials
  • RNA pool ( protocol 1)
  • 10× alkaline phosphatase buffer (New England Biolabs)
  • Calf alkaline phosphatase (New England Biolabs)
  • 25:24:1 phenol/chloroform/isoamyl alcohol saturated with 10 mM Tris⋅Cl, pH 8.0/1 mM EDTA (unit 2.1)
  • Chloroform
  • 3 M sodium acetate ( appendix 22)
  • 70% and 95% ethanol
  • 1 mg/ml blue‐dyed glycogen (GlycoBlue; Ambion)
  • 10× PNK buffer (New England Biolabs)
  • 10 U/µl T4 polynucleotide kinase (PNK; New England Biolabs)
  • 167 mCi/ml [γ‐32P]ATP (7000 Ci/mmol; ICN Biomedical or GE Healthcare Life Sciences)
  • 42° and 75°C water baths
  • Centri‐Sep Spin Columns (Princeton Separations)
NOTE: All solutions and buffers should be made with deionized water (18.2 MΩ resistivity), filtered through 0.2‐µm polyethersulfone (PES) membrane, and sterilized by autoclaving. Use sterile, disposable plasticware and micropipet tips with filter barriers where possible. See Chapter 4 introduction and unit 4.1 for guidelines on standard methods to protect against contaminating RNases. If ribonuclease contamination is found to occur, it may be eliminated by treating water with diethylpyrocarbonate (DEPC; see unit 4.1).

Support Protocol 2: Binding Assay with the End‐Labeled RNA Pool to Determine the Optimal Protein Concentration for Selection

  Materials
  • Radiolabeled RNA pool ( protocol 2)
  • Binding buffer (see )
  • Target protein
  • 65° to 75°C thermal cycler, water bath, or heat block
  • Minifold I Dot‐Blot System (Whatman)
  • Nylon transfer membrane (Hybond N+, GE Healthcare Life Sciences)
  • 0.45‐µm nitrocellulose transfer and immobilization membrane (BA85 Protran, Whatman)
  • Clean forceps or tweezers
  • PhosphorImager (GE Healthcare Life Sciences) and screen or X‐ray film and densitometer (also see appendix 3A)
  • Graphing software (e.g., SigmaPlot, Systat Software, or R Project)
  • Additional reagents and equipment for phosphor imaging or imaging using X‐ray film and densitometry ( appendix 3A)
NOTE: All solutions and buffers should be made with deionized water (18.2 MΩ resistivity), filtered through 0.2 µm polyethersulfone (PES) membrane, and sterilized by autoclaving. Use sterile, disposable plasticware and micropipet tips with filter barriers where possible. See Chapter 4 introduction and unit 4.1 for guidelines on standard methods to protect against contaminating RNases. If ribonuclease contamination is found to occur, it may be eliminated by treating water with diethylpyrocarbonate (DEPC; see unit 4.1).

Basic Protocol 2: Isolating a Functionally Enriched Pool of RNA

  Materials
  • RNA pool (see protocol 1)
  • Binding buffer (see )
  • Elution buffer (see recipe)
  • 3 M sodium acetate ( appendix 22)
  • 70% and 95% ethanol
  • 25:24:1 phenol/chloroform/isoamyl alcohol saturated with 10 mM Tris⋅Cl, pH 8.0/1 mM EDTA (unit 2.1), ice‐cold (optional)
  • Chloroform (optional)
  • Isopropanol (optional)
  • 65° to 75°C, 95°C. and 100°C heat blocks with appropriate bore sizes for the microcentrifuge tube
  • 13 mm Nuclepore Pop‐Top or Swin‐Lok Filter holders (Whatman)
  • 13‐mm, 0.45‐µm HAWP nitrocellulose disk filters (Millipore)
  • 5‐ml syringe
  • Vacuum manifold
  • Sterile forceps
NOTE: All solutions and buffers should be made with deionized water (18.2 MΩ resistivity), filtered through 0.2 µm polyethersulfone (PES) membrane, and sterilized by autoclaving. Use sterile, disposable plasticware and micropipet tips with filter barriers where possible. See Chapter 4 introduction and unit 4.1 for guidelines on standard methods to protect against contaminating RNases. If ribonuclease contamination is found to occur, it may be eliminated by treating water with diethylpyrocarbonate (DEPC; see unit 4.1).

Basic Protocol 3: Amplifying Selected Protein‐Binding RNA Species

  Materials
  • Selected RNA pool ( protocol 4)
  • TE buffer, pH 8.0 (see recipe), or RNase‐free water
  • SuperScript II reverse transcription kit (Invitrogen)
  • 20 and 200 µM 3′‐end primer
  • 4 mM dNTP mix (containing 4 mM each of dATP, dCTP, dGTP, and dTTP)
  • 10× PCR buffer (see recipe)
  • 20 µM 5′‐end primer
  • 5 U/µl Taq DNA polymerase (New England Biolabs)
  • 6× nondenaturing dye: 0.6% (w/v) bromphenol blue and 10× ethidium bromide in TBE buffer (see appendix 22 for TBE buffer)
  • NuSieve agarose (Cambrex)
  • 10 mg/ml ethidium bromide solution ( appendix 22)
  • TBE buffer ( appendix 22)
  • 3 M sodium acetate ( appendix 22)
  • 1 mg/ml blue‐dyed glycogen (GlycoBlue; Ambion)
  • 70% and 95% ethanol
  • Thermal cycler (e.g., BioRad DNAEngine with heated lid) and PCR tubes
  • Additional reagents and equipment for the polymerase chain reaction (Chapter 15), agarose gel electrophoresis (e.g., unit 2.6), and DNA sequencing (Chapter 7)
NOTE: All solutions and buffers should be made with deionized water (18.2 MΩ resistivity), filtered through 0.2‐µm polyethersulfone (PES) membrane, and sterilized by autoclaving. Use sterile, disposable plasticware and micropipet tips with filter barriers where possible. See Chapter 4 introduction and unit 4.1 for guidelines on standard methods to protect against contaminating RNases. If ribonuclease contamination is found to occur, it may be eliminated by treating water with diethylpyrocarbonate (DEPC; see unit 4.1).

Support Protocol 3: Assaying the Accumulation of Protein‐Binding RNA Species

  Materials
  • Pool of dsDNA after n rounds of selection ( protocol 5)
  • Binding buffer (see )
  • Target protein
  • 167 mCi/ml [α‐32P]ATP (7000 Ci/mmol; ICN Biomedical Inc or GE Healthcare Life Sciences)
  • Additional reagents and equipment for purifying a radiolabeled RNA pool (see protocol 1) and performing the filter binding assay (see protocol 3)
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Figures

Videos

Literature Cited

   Baskerville, S., Zapp, M., and Ellington, A.D. 1995. High resolution mapping of the human T‐cell, leukemia virus type 1 rex‐binding element by in vitro selection. J. Virol. 69:7559‐7569.
   Bell, S.D., Denu, J., Dixon, J.E., and Ellington, A.D. 1998. RNA molecules that bind to and inhibit the active site of a tyrosine phosphatase. J. Biol. Chem. 273:14309‐14314.
   Breaker, R. and Joyce, G.F. 1994. Emergence of a replicating species from an in vitro RNA evolution reaction. Proc. Natl. Acad. Sci. U.S.A. 91:6093‐6097.
   Breaker, R., Banerji, A., and Joyce, G.F. 1994. Continuous in vitro evolution of bacteriophage RNA polymerase promoters. Biochemistry 33:11980‐11986.
   Brody, E.N., Willis, M.C., Smith, J.D., Jayasena, S., Zichi, D., and Gold, L. 1999. The use of aptamers in large arrays for molecular diagnostics. Mol. Diagn. 4:381‐388.
   Chandra, S. and Gopinath, B. 2007. Methods developed for SELEX. Anal. Bioanal. Chem. 387:171‐182.
   Conrad, R.C., Giver, L., Tian, Y., and Ellington, A.D. 1996. In vitro selection of nucleic acid aptamers that bind proteins. Methods Enzymol. 267:336‐367.
   Cox, J.C., Rudolph, P., and Ellington, A.D. 1998. Automated DNA selection. Biotechnol. Prog. 14:845‐850.
   Ellington, A.D. and Szostak, J.W. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346:818‐822.
   Giver, L., Bartel, D., Zapp, M., Green, M., and Ellington, A.D. 1993. Selective optimization of the Rev‐binding element of HIV‐1. Nucleic Acids Res. 23:5509‐5516.
   Gold, L., Polisky, B., Uhlenbeck, O., and Yarus, M. 1995. Diversity of oligonucleotide functions. Annu. Rev. Biochem. 64:763‐797.
   Gopinath, S.C. 2007. Methods developed for SELEX. Anal. Bioanal. Chem. 387:171‐182.
   Guatelli, J., Whitfield, K., Kwoh, D., Barringer, K.J., Richman, D., and Gingeras, T.R. 1990. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. U.S.A. 87:1874‐1878.
   Irvine, D., Tuerk, C., and Gold, L. 1991. SELEXION: Systematic evolution of ligands by exponential enrichment with integrated optimization by non‐linear analysis. J. Mol. Biol. 222:739‐761.
   Jellinek, D., Lynott, C., Riata, D., and Janjic, N. 1993. High affinity RNA ligands to basic fibroblast growth factor inhibit receptor binding. Proc. Natl. Acad. Sci. U.S.A. 90:11227‐11231.
   Jhaveri, S., Olwin, B., and Ellington, A.D. 1998. In vitro selection of phosphorothiolated aptamers. Bioorg. Med. Chem. Lett. 8:2285‐2290.
   Keene, J.D. 1996. RNA surfaces as mimetics of proteins. Chem. Biol. 3:505‐513.
   Kramer, F.R., Mills, D.R., Cole, P.E., Nishihara, T., and Spiegelman, S. 1974. Evolution of in vitro sequence and phenotype of a mutant RNA resistant to ethidium bromide. J. Mol. Biol. 89:719‐736.
   Kulbachinskiy, A.V. 2007. Methods for selection of aptamers to protein targets. Biochemistry Mosc. 72:1505‐1518.
   Lato, S.M., Boles, A.R., and Ellington, A.D. 1995. In vitro selection of RNA lectins: Using combinatorial chemistry to interpret ribozyme evolution. Chem. Biol. 2:291‐303.
   Lehman, N. and Joyce, G.F. 1993. Evolution in vitro of an RNA enzyme with altered metal dependence. Nature 361:182‐185.
   Levisohn, R. and Spiegelman, S. 1969. Further extracellular Darwinian experiments with replicating RNA molecules: Diverse variants isolated under different selective conditions. Proc. Natl. Acad. Sci. U.S.A. 63:805‐811.
   Mills, D.R., Peterson, R.L., and Spiegelman, S. 1967. An extracellular Darwinian experiment with a self‐duplicating nucleic acid molecule. Proc. Natl. Acad. Sci. U.S.A. 58:217‐224.
   Shamah, S.M., Healy, J.M., and Cload, S.T. 2008. Complex target SELEX. Acc. Chem. Res. 41:130‐138.
   Stoltenburg, R., Reinemann, C., and Strehlitz, B. 2007. SELEX: A (r)evolutionary method to generate high‐affinity nucleic acid ligands. Biomol. Eng. 24:381‐403.
   Tuerk, C. and Gold, L. 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505‐510.
   Uphoff, K., Bell, S., and Ellington, A.D. 1996. In vitro selection of aptamers: The dearth of pure reason. Curr. Opin. Struct. Biol. 6:281‐288.
   Wlotzka, B. and McCaskill, J.S. 1997. A molecular predator and its prey: Coupled isothermal amplification of nucleic acids. Chem. Biol. 4:25‐33.
   Wright, C. and Joyce, G.F. 1997. Continuous in vitro evolution of catalytic function. Science 276:614‐617.
   Zichi, D., Eaton, B., Singer, B., and Gold, L. 2008. Proteomics and diagnostics: Let's get specific, again. Curr. Opin. Chem. Biol. 12:78‐85.
Key References
   Conrad et al., 1996. See above.
  The above papers also describe protocols for the selection of aptamers via both filter immobilization and other separation methods.
   Conrad, R.C., Bruck, F.M., Bell, S., and Ellington, A.D. 1998. In vitro selection of nucleic acid ligands. In Nucleic Acid‐Protein Interactions: A Practical Approach (W.J. Christopher, ed.) pp. 285‐315. Oxford University Press, New York.
   Gopinath, 2007. See above.
   Kulbachinskiy, 2007. See above.
   Fickert, H., Betat, H., and Hahn, U. 2004. Selection of Aptamers. In Evolutionary Methods in Biotechnology: Clever Tricks for Directed Evolution (S. Brakmann and A. Schwienhorst, eds.) pp. 65‐86. Wiley‐VCH, Weinheim, Germany.
   Stoltenburg et al., 2007. See above.
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